The present invention relates to the detection of ligand in solution. More particularly, the invention relates to a process and apparatus for performing homogeneous radioassays which do not require the separation step of conventional assays. Elimination of the separation step permits continuous monitoring of ligand level and promotes the possibility of automation of the assay.
Currently, a variety of processes are used to detect low levels of chemical materials or ligand in solution. Radioassays, particularly radioimmunoassays (RIA), are especially useful for biomedical applications because of their sensitivity and excellent reproducibility. RIAs have been developed which detect ligand levels of one nanogram (10.sup.-9 grams) with a reproducibility of better than one percent. While similar levels of reproducibility have been achieved with other assays, radioassays are the assay of choice because of their sensitivity.
The basis of all radioassays is the specificity of the reaction between a receptor molecule, e.g., an antibody, and the ligand, e.g., an antigen. High specificity means that the receptor will react with the ligand but is substantially unreactive with any other species. Generally, the greater the specificity of the ligand/receptor pair, the greater the sensitivity of the reaction.
While all radioassays depend on the ligand/receptor reaction, a number of radioassay procedures have been used to detect ligand concentration in samples. In every radioassay, there is a labelled or tagged species and an unlabelled species. Labelling or tagging means incorporating a radioactive atom into the molecule either by atomic substitution or by covelant bonding. In some cases, a macromolecule may be linked to a small ligand and the radioactive atom is incorporated into the macromolecule. The procedure chosen for the radioassay depends, in part, on the properties of the ligand of interest. If the ligand is easily tagged with a radioactive atom, competitive procedures whereby untagged ligand in the sample competes for receptor sites with tagged ligand are normally used. If the ligand is difficult to tag, the receptor, e.g., the antibody, may be tagged and the procedure chosen should include means for differentiating between free receptor molecules and ligand/receptor pairs.
Radioassays are further divided into solution assays and solid phase assays. In the solution assays, receptor molecules and ligands react in solution and the separation step differentially precipitates bound pairs from free species. Activated charcoal and ammonium sulfate precipitation are common methods of separating bound from free species in solution. The radioactivity level of either the precipitent or the solution can be counted in order to determine ligand concentration.
Solid phase radioassays were first described by Catt and his co-workers in 1967 (see Catt, et al, Solid Phase Radioimmunoassays; Nature 213: 825-827 (1967)). In solid phase assays, one species of the ligand/receptor pair is bound to a solid support while the other constitutes a free species. The bound member can be adsorbed onto the solid support, e.g., a test tube wall, or covalently bound on the support by a chemical reaction. In either case, a free species tagged with the radioactive atom is allowed to react with the bound member. In conventional techniques, the unreacted, labelled free species is separated from the bound, labelled free species and, preferably, the solid phase is counted for radioactivity. The resulting value normally is compared with a standard curve to determine the concentration of ligand in the sample. Conventional solid phase techniques include single and double antibody immunoassays. In single antibody techniques, the antibody is normally the bound species and labelled antigen competes with unlabelled antigen for the antibody binding sites. In double antibody techniques, antibody is bound to the solid support, labelled antibody is a free species in solution and the ligand forms a bridge or sandwich between bound antibody and labelled antibody.
Conventional radioassays are non-homogenous; that is, they require a separation step in order to function. This separation step is one of the causes of inaccuracy and other difficulties in automation of radioassays. To combat these problems, a number of homogenous assays have been developed using non-radioactive techniques. One such technique is the spin-immunoassay as described by Leute et al, Spinimmunoassay Technique of Opiate Narcotic in Urine and Saliva; J. Am. Med. Assoc., 221: 1231-1234 (1971). In a spinimmunoassay, the ligand is attached to a stable free radical whose concentration is determined by electron spin resonance (esr). Reaction of the spin-labelled ligand with an antibody diminishes the esr reading. The introduction of unlabelled ligand from a sample causes the spin-labelled ligand to be displaced from the antibody, increasing the esr of the solution and indicating the ligand concentration of the sample.
Another homogenous technique is the fluorescence polarization assay described by Haber et al, Polarization of Fluorescence as a Measure of Antigen-Antibody Interaction, Proc. Natl. Acad. Sci., USA 48: 1935-1942 (1962). In fluorescence polarization assays, the free radical bound to ligand in the spin-immunoassay is replaced with a dye that fluoresces only when the ligand is bound by an antibody. Unlabelled ligand reacts with antibody, displacing labelled ligand from the antibody and decreasing the fluorescence of the solution. The decrease in fluorescence is a measure of ligand concentration in the sample.
A further homogenous assay is the EMIT or Enzyme Multiplied Immunoassay Technique described by Bastiani in The EMIT System: A Commercially Successful Innovation; Antibiotics and Chemotherapy 26: 89-97 (1979). The EMIT system has the ligand bound to the enzyme proximate to the enzyme active site. Reaction with a antibody sterically blocks the enzyme activity. Addition of free ligand from the sample displaces antibody from the enzyme/ligand complex, causing an increase in enzyme activity indicative of the ligand concentration in the sample.
ELISA or enzyme linked immunosorbent assay, described by Enquall, et al, in Enzyme-Linked Immunosorbent Assay (ELISA), Quantitative Assay of Immunoglobulin G; Immunochem. 8: 871-879 (1971), is another nonradioassay technique which has been used with success. ELISA is the enzyme counterpart of previously described double antibody technique. Unlabelled antibody is bound to a solid support, e.g., a test tube wall, and reacts with a ligand having at least two reactive sites. The bound ligand binds the second antibody labelled with an enzyme and after a separation step, the bound enzyme concentration, indicative of ligand concentration, is measured.
The major problem with the homogenous enzyme assays is that the sensitivity of radioassays is better than the sensitivity of enzyme assays. Development of a homo-genous radioassay would be a step forward because a number of ligands of interest have very low concentration in biological fluids. It also should be noted that none of the homogenous assays described above is particularly well suited for in vivo ligand detection.
Accordingly, an object of the invention is to produce a homogenous radioassay having excellent sensitivity and reproducibility. Another object of the invention is to provide an apparatus for performing a homogenous radioassay. A further object is to provide a process and apparatus adapted for continous ligand assay. A still further object of the invention is to provide a process and apparatus adapted for in vivo determination of ligand concentration. Another object is to provide a process for ligand detection adaptable for automation. A further object is to provide a radioassay capable of use for ligand detection in a nonequilibrium mode.
These and other objects and features of the invention will be apparent from the following drawing and the description.